Proteolytic enzymes and their regulatory networks, including cofactors, activators, and endogenous inhibitors, are frequently dysregulated in tumors resulting in increased protease activities that contribute to progression of disease [1]. Manipulation of tumor-promoting proteases is a promising approach for the development of anti-tumor therapies [2,3]. While the targeting of proteases has been approached in several ways [4], prodrug-like protease substrates that are activated by overexpressed proteases are an extremely efficient approach to increasing selectivity and efficacy while reducing off-target effects [5].
Anthrax toxins requiring proteolytic activation have been engineered to target proteases overexpressed by tumor cells. Anthrax toxin is a cytotoxic pore-forming exotoxin secreted by Bacillus anthracis. Consisting of protective antigen (PrAg), lethal factor (LF), and edema factor (EF), the toxin (the combination of PrAg and LF and/or EF) causes cellular cytotoxicity through a well-characterized mechanism [6], whereas individually these proteins are non-toxic. PrAg binds to either of two cell-surface receptors, tumor endothelial marker-8 (TEM8, ANTXR1) and capillary morphogenesis gene-2 (CMG2, ANTXR2), of which CMG2 is expressed on nearly all cell types. PrAg (83 kDa) bound to its cell-surface receptor(s) is proteolytically cleaved and activated by the protease furin (FURIN) or furin-like proprotein convertases in an exposed flexible loop to generate an active C-terminal 63-kDa PrAg fragment.
The newly-generated 63-kDa PrAg fragment remains receptor bound and catalyzes the formation of a PrAg/receptor oligomer that presents docking sites to enable up to four molecules of LF or EF to bind and translocate into the cytosol of a cell, through an endosomal PrAg-formed pore, wherein LF/EF then have potent cytotoxic effects [7].
As a highly efficient protease-activated delivery system, PrAg can be engineered to deliver different payloads or co-factors into the cytosol [8-14]. Additionally, PrAg can be engineered to be activated specifically by proteases other than furin. Since furin is ubiquitously expressed, it is advantageous to narrow the cellular protease targets for drug delivery applications. Alteration of the furin protease cleavage site within PrAg to amino acid sequences recognized by either urokinase-type plasminogen activator (uPA, PLAU) [15], matrix metalloproteinase 2 (MMP2), or matrix metalloproteinase 9 (MMP9) [16] renders PrAg a potent uPA- or MMP2/9-activated prodrug that has been shown to target tumors that overexpress any of these proteases [17-26]. An engineered anthrax inter-complementing toxin has also been created that requires combined activation by these protease systems for function and killing of tumor cells [20,27].
While such uPA- or MMP2/9-activated prodrugs may be useful in some applications, in addition to their roles in tumor biology the uPA and MMP protease systems play leading roles in immune regulation and physiological tissue remodeling [4,28]. Therefore, while these engineered anthrax protein prodrugs are effective when used to target tumors in vivo, it is possible that paracrine association of the tumor-secreted proteases with other non-tumor cells in or near the tumor microenvironment could contribute to off-target effects of these toxin systems. Therefore, the use of existing protease-activated PrAg proteins is limited, and the development of new, targeted proteins is needed. The present application is directed to this and to other important goals.